Inhibitors of cell migration and shape changes by inhibiting cortactin and hs-1 medicated actin polymerization

ABSTRACT

The invention relates to methods for screening compounds for their ability to modulate actin polymerization. The invention is also related to compounds which modulate actin polymerization. More specifically, the invention is related to methods of screening compounds which modulate the interaction of cortactin or HS-1 with Arp2/3. The invention is also related to compounds which modulate the interaction of cortactin of HS1 with Arp2/3. Also provided are mutants of cortactin with decreased ability to interact with Arp2/3.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0001] Part of the work performed during development of this inventionutilized U.S. Government funds. The U.S. Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to methods for screening compounds fortheir ability to modulate actin polymerization. The invention is alsorelated to compounds which modulate actin polymerization.

[0004] More specifically, the invention is related to methods ofscreening compounds which modulate the interaction of cortactin or HS-1with Arp2/3. The invention is also related to compounds which modulatethe interaction of cortactin or HS1 with Arp2/3. Also provided aremutants of cortactin with decreased ability to interact with Arp2/3.

[0005] 2. Related Art

[0006] Amplification of the chromosome 11q13 constitutes an importantgenetic mechanism to progression of a subset of cancers, includingbreast cancer and head and neck carcinomas (Schuuring, E. D., et al.,Mol. Cell Biol. 13:2891-2898 (1993)). The amplification often results inoverexpression of cortactin, a filamentous actin (F-actin) associatedprotein. (Wu, H., et al., Mol. Cell Biol. 11:5113-5124 (1991); Zhan, X.,et al., J. Biol. Chem. 268:24427-24431 (1993)). While overexpression ofcortactin is usually correlated with poor prognosis (Schuuring, E., Gene159:83-96 (1995)), presumably due to enhanced metastasis, thebiochemical and biological functions of cortactin remain unclear.

[0007] Cortactin is a prominent substrate for several non-receptorprotein tyrosine kinases, including Src, Fer and Syk (Wu, H., et al.,Mol. Cell Biol. 11:5113-5124 (1991); Maruyama, S., et al., J Biol. Chem271:6631-6635 (1996); Kim, L., and Wong, T. W., J. Biol. Chem.273:23542-23548 (1998); Gallet, C., et al., J. Biol. Chem.274:23610-23616 (1999)). Tyrosine phosphorylation of cortactin is acommon cellular response to growth factors, stress, cell shrinkage, andcell injury mediated by reactive oxygen radicals (Zhan, X., et al., J.Biol. Chem. 268:24427-24431 (1993); Liu, M., et al., J. Biol. Chem.271:7066-7071 (1996); Kapus, A., et al., J. Biol. Chem. 274:8093-8102(1999); Li, Y., et al., (in press)). In vitro, tyrosine phosphorylationmediated by Src decreases the F-actin cross-linking activity ofcortactin. (Huang, C., et al., J. Biol. Chem. 272:3911-13915 (1997)).

[0008] Cortactin has a unique structure featured by six and one halftandem repeats of 37-amino-acid sequence and a carboxyl terminal SH3domain (FIG. 1A). Between the repeat and the SH3 domain there are aproline-rich sequence, an alpha helical region and three tyrosineresidues that are targeted by Src-related kinases (Huang, C., et al., J.Biol. Chem. 273:25770-25776 (1998)). In addition, cortactin contains anN-terminal domain, which is conserved across species and in HS1, acortactin-related protein that is exclusively expressed in thehematopoietic lineage (Kitamura, D., et al., Nucleic. Acids. Res.17:9367-9379 (1989)).

[0009] In cultured fibroblasts, cortactin is mainly distributed withincell leading edges such as lamellipodia and punctate-like structures(Wu, H., et al., Mol. Cell Biol. 11:5113-5124 (1991); Huang, C., et al.,J. Biol. Chem. 273:25770-25776 (1998)). In MDA-MB-231 cells, an invasivebreast cancer cell line, cortactin is associated with invadopodia,cortical structures that penetrate into and degrade extracellular matrixduring invasion (Bowden, E. T., et al., Oncogene 18:4440-4449 (1999)).

SUMMARY OF THE INVENTION

[0010] The invention relates to methods for screening compounds fortheir ability to modulate actin polymerization. The invention is alsorelated to compounds which modulate actin polymerization.

[0011] More specifically, the invention is related to screeningcompounds which modulate the interaction of cortactin or HS1 withArp2/3. The invention is also related to compounds which modulate theinteraction of cortactin or HS1 with Arp2/3.

[0012] The invention provides methods for treating diseases byadministering compounds which modulate actin polymerization and/ormodulate the interaction of cortactin or HS1 with Arp2/3. The inventionfurther provides compositions which modulate actin polymerization and/ormodulate the interaction of cortactin or HS1 with Arp2/3.

[0013] The invention also provides mutants of cortactin which havedecreased ability to interact with Arp2/3.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1. FIG. 1A shows a schematic presentation of the structuraldomains of cortactin. The domains that are targeted by Arp2/3 complex,F-actin and Src are labeled. The N-terminal amino acid sequence of humancortactin from 1 to 80 was compared to mouse, chicken and drosophilacortactin and human HS1. FIG. 1B shows a comparison of an acidicsequence in the N-terminus of murine cortactin with Arp2/3 complexbinding domains of other WASP-related proteins. The conserved Trp wasbolded.

[0015]FIG. 2. FIG. 2A shows the interaction of cortactin with Arp2/3complex in vivo. NIH 3T3 cell lysates were immunoprecipitated by eithera cortactin monoclonal antibody and protein G-Sepharose (lane 1) orprotein G-Sepharose alone (lane 2). The presence of Arp3, a subunit ofArp2/3 complex, in the cortactin pellets was detected by immunoblottingwith an Arp3 polyclonal antibody. The position of Arp3 at approximately50 kDa was indicated by an arrow (lane 1). (The major band shown inlanes 1 and 2 is background resulting from the cross-activity of theArp3 antibody to protein G-Sepharose). Lane 3, a positive control forArp3 (purified Arp2/3 complex). FIG. 2B shows the interaction ofrecombinant cortactin proteins with purified Arp2/3 complex byincubation of recombinant GST-cortactin with purified bovine Arp2/3complex. The resulting protein complexes were precipitated withglutathione-Sepharose and further immunoblotted with an Arp3 antibody.Lane 1, GST-CortΔ (1-38); lane 2, GST-cortactin; lane 3, GST-CortΔSH3;lane 4, GST; lane 5, GST-Cort(1-39); lane 6, GST-Cort(1-39); lane 7,GSTCort(D20G/D21A); and lane 8, GST-Cort(W22A).

[0016]FIG. 3. FIG. 3 lists a schematic summary of the analysis of theactivity of a series of cortactin mutants for Arp2/3 complex binding andstimulation of actin polymerization.

[0017]FIG. 4. FIG. 4A shows a time course of actin polymerizationmeasured by increase in fluorescence of polymerized pyrene actin,demonstrating the effect of cortactin on the actin polymerizationnucleated by the Arp2/3 complex. FIG. 4B shows the dose dependence ofcortactin mediated actin polymerization.

[0018]FIG. 5. Plasmids encoding cortactin-myc and CortΔ(1-69)-myc weretransiently transfected into MDA-MB-231 cells grown at log-phase andplated on fibronectin-coated glass cover slips. After two days oftransfection, the cells were fixed and stained with 9E-10 antibody forthe myc epitope or with FITC-phalloidin for F-actin and examined byconfocal microscopy.

[0019]FIG. 6. FIG. 6A shows a comparative histogram for thetransendothelial migration of MDA-MB-231 cells expressing cortactinmutants. The numbers of migrated cells after 20 h is shown. The numbersreflect the mean ± standard deviation of three independent experiments.FIG. 6B shows a comparative histogram for the migration of MDA-MB-231cells expressing cortactin mutants through a fibronectin-coatedpolycarbonate membrane. The numbers of migrated cells after 4 h isshown. The numbers reflect the mean ± standard deviation of fiveindependent experiments.

[0020]FIG. 7. FIG. 7 presents an analysis of bone metastases ofMDA-MB-231 cells in nude mice. FIG. 7A shows the average body weightafter 5 weeks for each test group. FIG. 7B shows the average number ofbone lesions found in the test groups as analyzed by radiography. Alldata shown represent the mean ± standard deviation (n=10).Representative radiography for each group is shown in FIG. 7C. Bonelesions at the joints between femurs and tibiae in high magnificationare indicated by arrows in the bottom panel of FIG. 7C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Cortactin, a filamentous actin associated protein and a prominentsubstrate of Src, is implicated in breast tumor progression via geneamplification at chromosome 11q13. However, the function of cortactinremains obscure. The inventors have discovered, for the first time, thatcortactin binds directly to Arp2/3 complex and activates Arp2/3 complexto promote actin polymerization in a manner independent of WASP-relatedproteins. The inventors have also discovered that the interaction ofcortactin with Arp2/3 complex occurs at an N-terminal domain rich inacidic amino acids. Mutations at a conserved amino acid sequence of DDWabolished the interaction with Arp2/3 complex as well as the activationof Arp2/3 complex. A cortactin mutant deficient in binding to Arp2/3complex fails to locate within membrane particulate structures ofMDA-MB-231 breast cancer cells. These findings indicate a role of theinteraction of cortactin with Arp2/3 complex in the actin polymerizationassociated with dynamic particulate structures, which have beenimplicated in tumor invasion and proteolysis of extracellular matrix.

[0022] The invention therefore provides a method of determining whethera test compound modulates the interaction of cortactin or HS-1 withArp2/3 comprising a. contacting monomeric or polymeric actin, cortactinor HS-1, and Arp2/3 with a test compound; and b. detecting an alterationin the interaction of cortactin or HS-1 with Arp2/3 when compared withthe interaction of cortactin or HS-1 with Arp2/3 in the absence of saidtest compound, wherein an alteration in the interaction of cortactin orHS-1 with Arp2/3 indicates that said test compound is a modulator of theinteraction of cortactin or HS-1 with Arp2/3.

[0023] As used herein, “test compound” means any compound, includingboth (a) compounds which are known to modulate the interaction ofcortactin or HS-1 with Arp2/3, and (b) compounds which are not known tomodulate the interaction of cortactin or HS-1 with Arp2/3.

[0024] The distribution pattern of cortactin within cells is verysimilar to that of Arp2/3 (Welch, M. D., et al., J. Cell Biol.138:375-384 (1997)), a protein complex composed of seven subunits thatplays a critical role in the nucleation of actin polymerization(Mullins, R. D., Curr. Opin. Cell Biol. 12:91-96(2000)). The fullactivity of Arp2/3 complex requires its association with WASP familyproteins, which are activated upon binding to membrane-associated smallGTPases such as Cdc42. (Aspenstrom, P., et al., Curr. Biol. 6:70-75(1996); Machesky, L. M., et al., Proc. Natl. Acad. Sci. USA 96:3739-3744(1999); Winter, D., et al., Curr. Biol 9:501-504 (1999); Yarar, D., etal., Curr. Biol 9:555-558 (1999); Egile, C., et al., J. Cell Biol.146:1319-1332 (1999); Rohatgi, R., et al., Cell 97:221-231 (1999)).

[0025] Within cells, cortactin exclusively associates with the corticalactin and is translocated to lamellipodia and membrane ruffles inresponse to stimulation. Cortactin's expression is nearly ubiquitous,except for in hematopoietic cells, where HS-1, a cortactin relatedprotein, is expressed. The failure to knock out cortactin in miceindicates that cortactin is essential for embryonic development.Conversely, overexpression of cortactin is a frequent event in cancersincluding breast cancer, head and neck squamous carcinoma. Subjects withhigh levels of cortactin expression have poor prognosis and have highermetastasized tumors.

[0026] To test the possibility that cortactin directly interacts withArp2/3 complex, the interaction of cortactin with Arp2/3 complex wasexamined by immunoprecipitation analysis. The lysate derived from NIH3T3 cells was precipitated with a cortactin-specific antibody followedby immunoblotting with an antibody against Arp3, a component in Arp2/3complex (Mullins, R. D., and Pollard, T. D., Curr. Opin. Struct. Biol.9:244-249 (1999)). The immunoblot analysis detected an Arp3-related bandonly in the pellet derived from the cortactin antibody but not from themock reaction (FIG. 2A). The apparent association of cortactin withArp2/3 complex might be due to an indirect interaction because bothcortactin and Arp2/3 complex can bind to F-actin. (Wu, H., and Parsons,J. T., J. Cell Biol. 120:1417-1426 (1993); Mullins, R. D., et al., Proc.Natl. Acad. Sci USA 95:6181-6186 (1998)). However, this possibility wasunlikely because recombinant cortactin was able to interact readily withpurified Arp2/3 complex in the absence of F-actin (FIG. 2B, lane 2).

[0027] By analyzing a series of deletion mutants in an in vitro actinpolymerization assay, it was determined that the N-terminus of cortactin(amino acids 1 to 38) was essential and sufficient to bind to Arp2/3complex (FIGS. 2 and 3). The N-terminus is remarkably conserved crossingspecies from human to Drosophila as well as in HS1 (FIG. 1A). Thissequence is rich in acidic amino acids and contains a Trp residue. Thesimilar amino acid composition is also found within the C-terminaldomains of WASP-related proteins (FIG. 1B). To test whether these acidicamino acids and the Trp residue were important for cortactin to bind toArp2/3 complex, Asp(20) and Asp(21) were mutated to Gly and Ala, andTrp(22) was mutated to Ala, respectively. As summarized in FIG. 4, bothmutations abolished the interaction with Arp2/3 complex. Because acidicamino acids and the Trp residue in the Arp2/3 complex binding domain ofWASP family proteins are important for their functions (Lechler, T., etal., J. Cell Biol. 148: 363-373 (2000)), this data suggests thatcortactin binds to Arp2/3 complex in a similar manner as WASP-relatedproteins.

[0028] The effect of cortactin on the activity of Arp2/3 complex wasexamined in more detail using the in vitro actin polymerization assaywith pyrene-labeled monomeric actin. Cortactin alone had no apparenteffect on the spontaneous actin polymerization, which is consistent withour previous report (Huang, C., et al., J. Biol. Chem. 272:3911-13915(1997)), whereas Arp2/3 complex alone increased moderately the rate ofactin polymerization (FIG. 4A). However, combination of Arp2/3 complexand cortactin reduced significantly the initial lag of actinpolymerization. The half time to reach a steady state of actinpolymerization in the presence of both Arp2/3 complex and cortactin wasapproximately 120 seconds, which was significantly less compared to 420seconds in the presence of Arp2/3 complex alone and 30 minutes withactin alone (Rohatgi, R., et al., Cell 97:221-231 (1999)). The activityof cortactin for actin polymerization is also dependent on theconcentration of Arp2/3 complex. The cortactin-mediated actinpolymerization came to a saturated level within approximately 200seconds in the presence of 500 nM Arp2/3 complex, whereas it required450 seconds to plateau in the presence of 250 nM Arp2/3 complex.Cortactin also activated Arp2/3 complex in a dose dependent manner witha maximal activity at 50 nM under the condition where the actinpolymerization was carried out in the presence of 2.5 μM actin and 200nM Arp2/3 complex (FIG. 4B).

[0029] The activity of cortactin for actin assembly requires its abilityto bind to Arp2/3 complex because mutants with deletion of sequencesessential for binding to Arp2/3 complex failed to activate Arp2/3complex (FIG. 3). However, binding to Arp2/3 complex itself seems notsufficient to stimulate actin polymerization. A peptide with the aminoacids from 1 to 39, which was able to bind efficiently to Arp2/3complex, was unable to stimulate actin polymerization (FIG. 3). Notably,a cortactin mutant containing only three repeats had 50% less affinityfor F-actin compared to the intact cortactin (Wu, H., and Parsons, J.T., J. Cell Biol. 120:1417-1426 (1993)) and exhibited only approximatelyhalf activity of the intact form to activate Arp2/3 complex-mediatedactin polymerization (FIG. 3). Thus, cortactin requires both activitiesbinding to F-actin and Arp2/3 complex to increase the rate of actinpolymerization. WASP family proteins require an actin-binding domain(WH2) as well as the acidic domain to activate Arp2/3 complex (Machesky,L. M., and Insall, R. H., Curr. Biol. 8:1347-1356 (1998)). However, therepeat domain of cortactin is distinct from the WH2 domain of WASP, thelatter has a strong affinity for monomeric actin but not forF-actin.(Machesky, L. M., and Insall, R. H., Curr. Biol. 8:1347-1356(1998); Miki, H., and Takenawa, T., Biochem. Biophys. Res. Commun.243:73-78(1998). In contrast, the repeat domain of cortactin binds alongthe side of F-actin (Ohoka, Y., and Takai, Y., Genes Cells 3:603-612(1998)). Therefore, it is possible that cortactin activates actinpolymerization by enhancing the association of Arp2/3 complex on theside of existing filaments (Machesky, L. M., et al., Proc. Natl. Acad.Sci. USA 96:3739-3744 (1999)). Because the N-terminal domain and therepeat domain of cortactin are highly conserved in HS1, HS1 has asimilar activity as cortactin to activate Arp2/3 complex-mediated actinassembly.

[0030] To evaluate the function of the interaction of Arp2/3 complexwith cortactin within cells, the distribution of cortactin in humanMDA-MB-231 breast cancer cells was analyzed. MDA-MB-231 cells containfew stress fibers as analyzed by phalloidin staining (FIG. 5). Instead,they deposit most F-actin into large particulate structures that areassociated with membrane. Within these particulate structures,endogenous cortactin (data not shown) or a wild-type cortactin tagged bymyc epitope is accumulated (FIG. 5). In contrast to wild-type cortactin,a cortactin mutant with deletion of amino acids from 1 to 69 diffusedwithin the cytoplasm and failed to localize within the F-actin-enrichedparticulate structures. Previous studies have demonstrated thatcortactin is accumulated within invadopodium, a cortical particulateprotrusion on the ventral side of cells that is implicated in thedegradation of and penetration into the extracellular matrix, whenMDA-MB-231 cells are plated on extracellular matrix (Bowden, E. T., etal., Oncogene 18:4440-4449 (1999)). Interaction with Arp2/3 complex istherefore required for the association of cortactin with F-actinenriched particulate protrusions of breast cancer cells.

[0031] Actin and cortactin-enriched particulate structures are alsoabundant in other types of cells such as osteoclasts (Hiura, K., et al.,Cell Motil. Cytoskeleton 30:272-284 (1995)). In osteoclasts, thesestructures are called as podosomes and used to seal off a region of bonewhere resorption of bone occurs (Blair, H. C., et al., Science245:855-857 (1989)).

[0032] Thus, Arp2/3 complex and cortactin mediated actin polymerizationplay a general role in the formation of dynamic cellular particulatestructures by which cells interact with either extracellular matrix orother cells where extensive proteolysis takes place. It is also knownthat cortactin binds to several cellular proteins via either itsphosphorylated tyrosine residues or the SH3 domain (Okamura, H., andResh, M. D., J. Biol. Chem. 270:26613-26618 (1995); Takemoto, Y., etal., EMBO J. 14:3403-3414 (1995); Stevenson, B. R., et al., J. CellBiol. 103:755-766 (1986); Ohoka, Y., and Takai, Y., Genes Cells3:603-612 (1998); Du, Y., et al., Mol. Cell Biol. 18:5838-5851 (1998)).Hence, cortactin may link those proteins to the actin polymerization.

[0033] In light of the expression profile of cortactin, the discovery ofthe interaction of cortactin with Arp2/3 complex has a broadimplications in other pathological processes in addition to tumormetastasis and osteoporosis. Abundant cortactin is also found withinneural fibers, epithelial cells and megakaryocytes and platelets.Furthermore, tyrosine phosphorylation of cortactin is dramaticallyelevated in response to platelet agonists and reactive oxygen species,which play important roles in the development of thrombosis,hypertension, and atherosclerosis. Because these processes areintimately associated with the cytoskeletal changes, targeting atcortactin provides a new therapeutical approach to compromise thesediseases.

[0034] In order to examine the potential role of cortactin inpathogenesis, several studies were initiated on MDA-MB-231 breast cancercells that overexpress cortactin mutants, which included examining theirpotential for transendothelial invasion (De Bruyn, P. P., et al., JMorphol. 133:417-437 (1971)). Invasion is a necessary metastatic eventfor tumor cells to migrate from the blood circulation to new tissues andrequires the ability of tumor cells to change shape and invade acrossthe endothelial lining between blood vessel and peripheral tissues. Inthis example, transendothelial invasion of tumor cells was evaluated.MDA-MB-231 cells expressing cortactin mutants were seeded over confluenthuman bone marrow endothelial cells (HBMEC) grown in the top chamber ofTranswell, a modified Boyden chamber apparatus. After 20 h, the cellsthat migrated through the endothelial layer on the membrane on thebottom chamber were inspected by fluorescence microscopy. The cellularmigration was analyzed by counting three high power field digital imagesrandomly taken at 200× magnification. The average of cell number and thestandard derivation were calculated based on duplicated experiments. Asshown in FIG. 6A, cells expressing the wild-type cortactin(wt-cortactin) increased transendothelial invasion about 40% compared tothe control cells expressing the vector only, whereas invasion wasdecreased by 45% in cells expressing the cortactin mutant deficient intyrosine phosphorylation (Cort_(F421F466F482)).

[0035] To further address the role of the interaction of cortactin withArp2/3 complex in cell migration, a cortactin mutant was constructedcontaining a deletion of amino acids from 1 to 34 (Cortdel(1-34)), theregion that is required for cortactin association with Arp2/3 complex.MDA-MB-231 cells expressing this cortactin mutation under the control ofMGIN virus were evaluated for their motility using Transwell apparatus.Cells were seeded on a fibronectin-coated polycarbonate membrane insertin a Transwell and incubated in DMEM containing 10% FBS. After 4 h, thecellular migration was analyzed by counting five high power fieldsdigital images randomly taken at 200× magnification. The average of cellnumber and the standard derivation were calculated based on duplicatedexperiments. The migration of was compared to the cells expressingwt-cortactin and the cells expressing the vector only. As shown in FIG.6B, the cells expressing the mutation (Cortdel(1-34)) displayed 60% lessmigration through the membrane than the control cells. Thus, disruptionof the interaction between cortactin and Arp2/3 complex effectivelymodulated cell migration. Cortactin mutant breast cancer cells deficientin either tyrosine phosphorylation (FIG. 6A) or Arp2/3 binding (FIG. 6B)demonstrated impaired motility as seen in Example 7.

[0036] In order to examine whether cortactin mutants could interferewith tumor metastasis, an animal model for bone metastasis was employedin which MDA-MB-231 cells expressing cortactin mutants were injectedinto the left ventricles of nude mice (Mbalaviele, G. et al., CancerRes. 56:4063-4070 (1996)). Five (5) weeks following the injection, theanimals bearing wt-cortactin cells developed apparent cachexia (loss ofmuscle, fat, and body weight). These animals had an average body weight25% less than that of control animals injected with cells expressing theviral vector only (FIG. 7A). In contrast, the average body weight ofmice bearing Cort_(F421F466F482) was about 20% higher than that of thecontrol animals. Tumors that had metastasized into bone were examined byX-ray radiography, and revealed tumor-induced bone lesions asradiolucent foci (FIG. 7C). As summarized in FIG. 7B, the number ofmetastatic lesions present was approximately 15% of those seen in thecontrol group. Further, mice bearing wt-cortactin cells developed anaverage of 7 tumors per animal, whereas the mice injected withCort_(F421F466F482) cells showed much less potential for bone metastasiswith only one tumor per animal. This is consistent with previousfindings that the mutation acts in a dominant negative manner withincells (Li, Y., et al., J Biol. Chem. 275:37187-93 (2000)), a resultwhich confirms the function of cortactin in tumor metastasis.

[0037] The invention provides methods for screening for compounds whichinhibit or activate actin polymerization. The invention is also directedto methods for screening for compounds which modulate the interactionbetween cortactin or HS-1 and Arp2/3. As used herein, it is understoodthat the term “cortactin” encompasses polypeptides which are at least80% identical, 90% identical, preferably 95% identical, 96% identical,97% identical, 98% identical or 99% identical to the amino acid sequenceof cortactin. As used herein, it is understood that the term “HS-1”encompasses polypeptides which are at least 80% identical, 90%identical, preferably 95% identical, 96% identical, 97% identical, 98%identical or 99% identical to the amino acid sequence of HS-1.

[0038] As used herein, “an inhibitor of the interaction of cortactin orHS-1 with Arp2/3” is a compound which weakens or blocks the interactionof cortactin or HS-1 with Arp2/3. As used herein, “an enhancer of theinteraction between cortactin or HS-1 and Arp2/3” is a compound whichpromotes or strengthens the interaction of cortactin with Arp2/3.

[0039] The interaction between cortactin or HS-1 and Arp2/3 in thepresence of the compound which is being screened can be assayed by anyof the methods and techniques known to those skilled in the art. Suchmethods include the pull-down assay (Uruno et al., Nature Cell Biology3:259-266 (2001)), immunofluorescence, immunoprecipitation, gelmigration analysis, radioisotopic tracer analysis and enzyme-linkedimmunosorbent assay (ELISA). A preferred assay technique is thepull-down assay.

[0040] Actin exists in three forms within cells: G-actin (monomericactin), stress fibers (cross-linked actin filaments) and cortical acin(branched actin filaments). In response to extracellular signals, actinis rapidly polymerized from G-actin into cortical actin filaments.Because cell movement is the reflection of the extension onlamellipodia, the rate of the polymerization of cortical actinconstitutes an important limit step for cell migration and cell shapechanges. Accordingly, a preferred assay technique for use in the presentinvention is placing fluorescent labeled monomeric actin inpolymerization buffer (10 mN Tris buffer, pH 7.5, containing 1 mM EGTA,0.1 mM CaCl₂, 0.5 mM dithiothreitol, and 0.2 mM ATP), then initiatingpolymerization by adding 50 mM KCl and 1 mM MgCl₂. Polymerization can bemeasured, for example, by monitoring the increase in fluorescence of thefluorescent label.

[0041] As used herein, “fluorescent label” includes any moleculesuitable to enable the detection of the interaction between cortactin orHS-1 and Arp2/3 in the presence of a compound which is being screened.An example of such a fluorescent label is pyrene. Other suitablefluorescent labels will be apparent to one of ordinary skill in the art.An increase in the rate of fluorescence (arbitrary units (au) per unittime) or intensity of fluorescence in the presence of cortactin or HS-1,Arp2/3 and the screened compound can be an indication that the screenedcompound is an enhancer of the interaction between cortactin or HS-1 andArp2/3. A decrease or absence of fluorescence in the presence ofcortactin or HS-1, Arp2/3 and the screened compound can be an indicationthat the screened compound is an inhibitor of the interaction betweencortactin or HS-1 and Arp2/3.

[0042] As used herein, “administering” may be read so as to encompassall methods of delivery of a pharmaceutic agent known to one of skill inthe art, also to encompass compounds which, when metabolized ordegraded, produce the desired effect.

[0043] The invention is also drawn to compounds which modulate theinteraction of cortactin or HS-1 with Arp2/3. The invention is alsodrawn to cortactin mutants or HS-1 mutants which are deficient inbinding to Arp2/3.

[0044] An example of a method of screening for a cortactin inhibitor isexamination of co-localization of Arp2/3 and cortactin proteins inmammalian cells. Arp2/3 and cortactin proteins can be in situ detectedwithin cells by immunofluorescent staining using antibodies specificallyagainst Arp2/3 and cortactin. Alternatively, mammalian cells can betransfected with plasmids or viral vectors carrying DNA fragmentsencoding cortactin proteins tagged with epitopes such as GFP, myc or HA.Thus, the subcellular localization of cortactin can be determined bystaining cells with fluorescent-labeled antibodies against theseepitopes.

[0045] Examples of inhibitors of actin polymerization encompassed by thepresent invention include compounds which bind to F-actin, but notArp2/3, and compounds which bind to Arp2/3, but not F-actin. Suchcompounds may be determined empirically by one skilled in the art usingroutine screening methods.

[0046] Examples of inhibitors of the interaction of cortactin or HS-1and Arp2/3 encompassed by the present invention include compounds whichinteract with the N-terminal domain of cortactin or HS-1, which is richin acidic amino acids. Such compounds may be determined empirically oneby skilled in the art using routine screening methods.

[0047] Examples of enhancers of actin polymerization encompassed by thepresent invention include compounds which bind to F-actin, but notArp2/3, and compounds which bind to Arp2/3, but not F-actin. Suchcompounds may be determined empirically by one skilled in the art usingroutine screening methods.

[0048] Examples of enhancers of the interaction of cortactin or HS-1 andArp2/3 encompassed by the present invention include compounds whichinteract with the N-terminal domain of cortactin or HS-1, which is richin acidic amino acids. Such compounds may be determined empirically oneby skilled in the art using routine screening methods.

[0049] Compounds which inhibit actin polymerization and/or compoundswhich inhibit the interaction of cortactin or HS-1 with Arp2/3 can beemployed to treat tumor metastasis. Cortactin is often overexpressed inmalignant tumors and in accumulated in invasopodia, a structure forcells to penetrate into the extracellular matrix.

[0050] Compounds which inhibit of actin polymerization and/or compoundswhich inhibit the interaction of cortactin or HS-1 with Arp2/3 can beemployed to treat osteoporosis. Cortactin is enriched in the membraneprotrusions of osteoclasts, which are used for the resorption of bonetissues. It is also known that mice deficient in Src can developosteoporosis symptoms because they fail to form those protrusionstructures in osteoclasts. Formation of these structures is known torequire actin polymerization, which requires the function of cortactin.Interruption of the activity of cortactin will inhibit the activity ofosteoclasts to resorb bone tissues and ultimately compromiseosteoporosis.

[0051] Compounds which inhibit actin polymerization and/or compoundswhich inhibit the interaction of cortactin or HS-1 with Arp2/3 can beemployed to inhibit angiogenesis. Recent studies have shown a criticalrole of Src in angiogenesis, which requires the migration of endothelialcells and degradation of extracellular basement membrane. Inhibition ofcortactin will inhibit angiogenesis as well.

[0052] Compounds which inhibit actin polymerization and/or compoundswhich inhibit the interaction of cortactin or HS-1 with Arp2/3 can beemployed to treat thrombosis. Cortactin is highly expressed in plateletsand megakaryocytes. Tyrosine phosphorylation of cortactin in response toplatelet agonists is a well-characterized and profound phenomenon, whichis believed to promote actin cytoskeleton reorganization. Becausetyrosine phosphorylation of cortactin occurs prior to the activation ofintegrins on the surface of platelets, the cortactin-mediated actinreorganization may play an important role for platelet aggregation.Inhibition of cortactin is expected to decrease the response ofplatelets to these agonists.

[0053] Compounds which enhance actin polymerization and/or compoundswhich enhance the interaction of cortactin or HS-1 with Arp2/3 can beemployed to improve wound healing. Enhancement of cortactin's activityis expected to enhance cell migration, which is essential for healing ofulcers or wounds.

[0054] Compounds which enhance actin polymerization and/or compoundswhich enhance the interaction of cortactin or HS-1 with Arp2/3 can beemployed to promote neurogenesis. Cortactin is enriched in the neuronfibers where actin polymerization is a key event in the neurite grown.The drugs that enhance the activity of cortactin should promote thegrowth of neurons and may potentially compromise neurondegeneration-related diseases such as Alzheimer's disease.

[0055] In accordance with another embodiment of the present inventionthere are provided methods of treating and processes of administeringcompounds to a host which would enhance actin polymerization and/orenhance the interaction of cortactin or HS-1 with Arp2/3 which areuseful in stimulating wound healing and neurogenesis. Provided by thepresent invention is a method of promoting wound healing andneurogenesis in a mammal in need thereof comprising administering to amammal in need thereof a therapeutically effective amount of a compoundwhich would enance actin polymerization and/or enhance the interactionof cortactin or HS-1 with Arp2/3.

[0056] In accordance with still another embodiment of the presentinvention there are provided methods of treating a subject in need oftreatment comprising administering to a subject a therapeuticallyeffective amount of a compound which would inhibit actin polymerizationand/or inhibit the interaction of cortactin or HS-1 with Arp2/3 whichare useful in preventing or treating tumor metastasis, osteoporosis,angiogenesis and thrombosis. Preferably, the tumor metastasis is tumormetastasis of breast cancer cells. Preferably, the subject in need oftreatment is a mammal. Preferably, the mammal is a human.

[0057] Also provided by the present invention is a mutant of cortactin,wherein the mutant confers to tumor cells about 0% to about 50%decreased ability to invade transendothelial cells when compared to thewild type cortactin derived from breast cancer cells. Preferably, saidmutant confers to tumor cells from about 5% to about 45% decreasedability to invade transendothelial cells when compared to the wild typecortactin derived from breast cancer cells. More preferably, said mutantconfers to tumor cells from about 10% to about 35% decreased ability toinvade transendothelial cells when compared to the wild type cortactinderived from breast cancer cells. Even more preferably, said mutantconfers to tumor cells from about 20% to about 25% decreased ability toinvade transendothelial cells when compared to the wild type cortactinderived from breast cancer cells.

[0058] The compounds which modulate actin polymerization and/or enhancethe interaction of cortactin or HS-1 with Arp2/3 can be employed incombination with any of the pharmaceutical carriers known to thoseskilled in the art. Such compositions generally comprise atherapeutically effective amount of the compound and a pharmaceuticallyacceptable carrier or exipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation employed should suit the modeof administration. Suitable formulations may be determined empiricallyby one skilled in the art.

[0059] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the compounds of the present invention may be employed inconjunction with other therapeutic compounds.

[0060] The pharmaceutical compositions may be administered by any of themethods and routes known to those skilled in the art, including topical,intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal. The pharmaceutical compositions are preferably administeredin an amount of at least about 10 mg/kg body weight and in most casesthey will be administered not in excess of about 8 mg/Kg body weight perday. Most preferably, the dosage is from about 1 mg/Kg to about 10 mg/Kgbody weight daily, taking into account the routes of administration,symptoms, etc.

EXAMPLES Example 1 Preparation of Arp2/3 Complex

[0061] Bovine Arp2/3 complex was purified by a modified procedure asdescribed previously (Egile, C., et al., J. Cell Biol. 146:1319-1332(1999)). Briefly, 100 g of frozen bovine brain was minced with a Waringblender in 100 ml of 20 mM Tris, pH 8.0, containing 100 mM NaCl, 5 mMMgCl₂, 5 mM EGTA and 1 mM DTT (buffer Q) supplemented with 50 μg/mlphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin and 1 μg/ml aprotinin.The minced tissue was further homogenized by a Dounce homogenizer andclarified by centrifugation at 40,000×rpm for 60 min at 4° C. Thesupernatant was subjected to chromatography in a 100 ml Sepharose Qcolumn equilibrated with buffer Q. The flow-through containing Arp2/3complex was collected, supplemented with 0.1 mM ATP and fractionated ona GST-N-WASP glutathione Sepharose column equilibrated with 50 mM Tris,pH 7.5, containing 25 mM KCl, 1 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT and 0.1mM ATP (buffer B). After washing with 0.2 M KCl in buffer B, the Arp2/3complex was eluted with buffer B containing 0.2 M MgCl₂. The protein wasthen dialyzed against buffer B and concentrated by a Centriprep 10cartridge. The concentrated Arp2/3 complex was stored in buffer Bcontaining 30% glycerol at −80° C. Protein concentration was determinedby the Bradford method (Bio-Rad protein Assay; Bio-Rad) using bovinegamma globulin as a standard. Concentrations at mg/ml were converted tomolar concentrations based on the equivalence of 1 mg/ml to 18 μMcortactin, 23 μM actin, and 4.8 μM Arp2/3 complex.

Example 2 Analysis of Interaction of Arp2/3 Complex and Cortactin

[0062] Glutathione Sepharose conjugated with GST or GST-cortactin wasmixed with 10 pmol of purified Arp2/3 complex in 100 μl of 50 mM Tris,pH 8.0, containing 150 mM NaCl, and 1% Triton X 100 (buffer A), andincubated for 2 h at 4° C. on a rotating wheel. The beads were rinsedthree times with buffer A followed by boiling in 2×SDS sample buffer.Arp2/3 complex was detected by immunoblotting using a polyclonal Arp3antibody after resolving by SDS-polyacrylamide gel electrophoresis. Datais shown in FIG. 2.

Example 3 In Vivo Interaction Assays

[0063] NIH 3T3 cells were lysed in buffer A supplemented with a proteaseinhibitor cocktail, and clarified by centrifugation at 14,000×rpm for 10min at 4° C. The supernatants were immunoprecipitated with 20 μl ofprotein G-Sepharose with and without 2 μg of cortactin monoclonalantibody 4F11. The resulting immunoprecipitates were washed twice withbuffer A, dissolved in 2×SDS sample buffer and subjected to SDS-PAGEfollowed by immunoblotting with Arp3 antibody, shown in FIGS. 2A and 2B.

Example 4 Construction of Cortactin Mutants

[0064] Deletion cortactin mutants, CortΔ(1-23), CortΔ(1-38),CortΔ(1-68), Cort(1-39), Cort(1-69), and Cort(1-80), were generated byPCR mutagenesis using the murine cortactin cDNA (pXZ112) as a template(Zhan, X., et al., J. Biol. Chem. 268:24427-24431 (1993)) with PfuTurbopolymerase (Stratagene). Point mutants Cort(W22A) and Cort(D20GD21A) aregenerated by site-directed mutagenesis using the Quickchangesite-directed mutagenesis kit (Stratagene). All the constructs usedherein, except for the three-repeat deletion mutant (pCH81), were firstgenerated as the EcoRI-Sal 1 fragments then further, sub-cloned into thepUC19 plasmid. The resulting plasmids served as the master plasmids,from which the inserts were excised and further cloned into either thepGEX4T-2 (Amersham Pharmacia) for bacterial GST-fusion proteinexpression, or the pCMV-Tag5B plasmid (Stratagene) for mammalianexpression of C-terminal myc-tagged proteins. The inserts in the masterplasmids were verified by DNA sequencing. Comparison of exemplarymutants generated by this procedure may be seen at FIG. 3. Thereferenced numbers used are based on the amino acid sequence of murinecortactin (Zhan, X., et al, J. Biol. Chem. 268:24427-24431 (1993)).

Example 5 Actin Polymerization

[0065] Pyrene-labeled monomeric actin at concentration of 2.8 μM wasincubated at 22° C. in polymerization buffer (10 mM tris buffer, pH 7.5,containing 1 mM EGTA, 0.1 mM CaCl₂, 0.5 mM dithiothreitol, and 0.2 mMATP). Polymerization was initiated by adding KCl and MgCl₂ to finalconcentrations of 50 mM and 2 mM respectively, and monitored bymeasuring the increase in pyrene fluorescence using a LS50Bspectrophotometer (Perkin Elmer) with filters for excitation at 365 nmand emission at 407 nm (15 nm and 10 nm band width, respectively). Datashown in FIG. 4 are the representatives of 24 experiments with identicalresults.

Example 6 Fluorescence Microscopy

[0066] MDA-MB-231 cells were transiently transfected with plasmidsencoding cortactin-myc proteins with a Superfect transfection kitaccording to the manufacturer's protocol. The cells were plated on aglass cover slip pre-coated with fibronectin. After 24 hours, cells werefixed with 10% formaldehyde in PBS for 10 minutes and permeabilized with0.5% Triton X-100 for 5 minutes. The treated cells were incubated for 1h with a monoclonal anti-myc antibody (9E10) at the concentration of 2μg/ml in PBS containing 5% BSA and then with a rhodamine-conjugated goatanti-mice antibody at 14 μg/ml in the same buffer for 30 minutes. ForF-actin staining, fluorescein isothiocyanate-labeled phalloidin wasadded at a final concentration of 1 μM. Between each step, three-timewashes with PBS were performed. The stained cells were finally mountedand examined under an Olympus Fluoroview confocal microscope using0.60×/1.4 N.A. lens as shown in FIG. 5.

Example 7 Transendothelial Invasion and Cell Migration

[0067] Transendothelial invasion of MDA-MB-231 cells was analyzed basedon a modified method as described (Okada,T., et al., Clin. Exp.Metastasis 12:305-314 (1994)). Briefly, human bone marrow endothelialcells (3×10⁵) were plated on a fibronectin-coated polycarbonate membraneinsert (with 6.5 mm in diameter and 8.0 μm pores) in a Transwellapparatus. After cells reached confluence, MDA-MB-231 cells expressingwt-cortactin variants were trypsinized and resuspended in DMEMcontaining 10% FBS. The suspended cells (3×10⁴) were seeded in amonolayer of endothelial cells and incubated for 20 h at 37° C. in a CO₂incubator.

[0068] For cell migration analysis, tumor cells were directly plated onfibronectin coated polycarbonate insert and incubated for 4 hours. Afterincubation, the insert was washed with PBS. The cells on the uppersurface of the insert were removed by wiping with a cotton swab. Thecells migrated to the lower surface of the insert were fixed with 3.7%formaldehyde and subjected to fluorescence microscopic inspection. Greencells were counted based on five HPF digital images randomly taken at200× magnification. The average of cell number and the standardderivation were calculated based on duplicated experiments. Results areshown in FIG. 6.

Example 8 Intracardiac Injections of MDA-MB-231 Cells into Nude Mice

[0069] Subconfluent MDA-MB-231 cells were maintained in DMEM containing10% FBS 24 h before injection. The cells were trypsinized, immediatelysuspended in DMEM containing 0.2 mg/ml soybean trypsin inhibitor, andwashed twice with PBS. The washed cells were finally resuspended in coldPBS at a density of 2.5×10⁶ cells/ml on ice. Female BALB/c-nu/nu mice atages of 4 to 5 weeks (National Cancer Institute, Frederick, Md.) wereanesthetized. MDA-MB-231 cells (5×10⁵) expressing wild-type or mutantcortactin protein were injected into the left ventricle of the animals.For each cell sample, 10 animals were analyzed. At 5 weeks afterinjection, the body weights of the mice were measured and the micefurther examined by X-ray radiograph. FIG. 7 presents an analysis ofbone metastases of MDA-MB-231 cells in the subject animals. All datashown represent the mean ± standard deviation (n=10). A representativeradiograph is also shown. The bone lesions at the joints between femursand tibiae in high magnification are indicated by arrows in the bottompanel.

[0070] All patents and publications referred to herein are herebyexpressly incorporated in their entirety by reference.

[0071] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

What is claimed is:
 1. A method of determining whether a test compoundmodulates the interaction of cortactin with Arp2/3 comprising a.contacting monomeric or polymeric actin, cortactin, and Arp2/3 with atest compound; and b. detecting an alteration in the interaction ofcortactin with Arp2/3 when compared with the interaction of cortactinwith Arp2/3 in the absence of said test compound, wherein an alterationin the interaction of cortactin with Arp2/3 indicates that said testcompound modulates the interaction of cortactin with Arp2/3.
 2. A methodof determining whether a test compound modulates the interaction of HS-1with Arp2/3 comprising a. contacting monomeric or polymeric actin, HS-1,and Arp2/3 with a test compound; and b. detecting an alteration in theinteraction of HS-1 with Arp2/3 when compared with the interaction ofHS-1 with Arp2/3 in the absence of said test compound, wherein analteration in the interaction of HS-1 with Arp2/3 indicates that saidtest compound modulates the interaction of HS-1 with Arp2/3.
 3. Themethod of claim 1 or claim 2, wherein said detecting an alteration inthe interaction of said cortactin or said HS-1 with Arp2/3 isaccomplished by an assay selected from the group comprising thepull-down assay, immunofluorescence, immunoprecipitation, gel migrationanalysis, radioisotopic tracer analysis and enzyme-linked immunosorbentassay (ELISA).
 4. The method of claim 1 or 2, wherein said monomeric orpolymeric actin is labeled with a fluorescent compound.
 5. A compoundwhich modulates the interaction of cortactin with Arp2/3, as indicatedby the method of claim 1 or claim
 2. 6. A method of treating tumormetastasis, osteoporosis, angiogenesis, and thrombosis in a subject inneed thereof, comprising administering to a subject in need thereof atherapeutically effective amount of the compound of claim
 5. 7. Themethod of claim 6, wherein said tumor metastasis is tumor metastasis ofbreast cancer cells.
 8. The method of claim 6, wherein said subject is amammal.
 9. The method of claim 8, wherein said mammal is a human.
 10. Amethod of promoting wound healing and neurogenesis in a mammal in needthereof comprising administering to a mammal in need thereof atherapeutically effective amount of the compound of claim
 5. 11. Amutant of cortactin, wherein said mutant confers to tumor cells aboutfrom 0% to 50% decreased ability to invade transendothelial cells whencompared to the wild type cortactin derived from breast cancer cells.12. The mutant of claim 11, wherein said mutant contains deletions ofamino acids 1 to
 34. 13. The mutant of claim 11, wherein said mutant isdeficient in tyrosine phosphorylation.
 14. A method of treating tumormetastasis, osteoporosis, angiogenesis, and thrombosis in a subject inneed thereof, comprising administering to a subject in need thereof themutant of claim
 11. 15. The method of claim 14, wherein said tumormetastasis is tumor metastasis of breast cancer cells.
 16. The method ofclaim 14, wherein said subject is a mammal.
 17. The method of claim 16,wherein said mammal is a human.
 18. A method of promoting wound healingand neurogenesis in a mammal in need thereof comprising administering toa mammal in need thereof the mutant of claim 11.